Project Summary Alcoholic liver disease (ALD) encompasses a spectrum of injury, ranging from simple steatosis to serious cirrhosis. Obesity and excess body weight is strongly associated with the severity of ALD. Ethanol-induced oxidative stress is a major mechanism by which ethanol induces liver injury. Increased CYP2E1 and reduced SOD1 activity leads to increased levels of superoxide in ethanol-fed conditions. Although oxidative stress appears to be a common mechanism linking obesity with ALD, the role of oxidative stress initiated by superoxide in modulating the pathogenesis of obesity-linked ALD is unclear. Moreover, the potential role of antioxidants in modulating the progression of ALD in obesity remains largely unknown. We recently reported that nanoformulated copper/zinc superoxide dismutase (nanoSOD) attenuates adipose tissue inflammation and non-alcoholic fatty liver disease (NAFLD) in obesity. Our preliminary data show that a combination of ethanol and linoleic acid (LA), a dietary polyunsaturated fatty acid, induced oxidative stress in primary hepatocytes and CYP2E1 over-expressing HepG2 cells. Our data also show that ethanol and LA in combination evoked profound increase in oxidative stress in steatotic hepatocytes derived from HF diet-fed rats, indicating that the presence of NAFLD can exacerbate ethanol-induced liver injury. We also provide evidence that primary hepatocytes derived from chronic ethanol-fed rats displayed increased oxidative stress compared to control which was attenuated by nanoSOD. Finally, we provide evidence for a possible role of AMPK signaling in mediating the anti-steatotic effects of nanoSOD. Together, these data suggest that delivery of SOD to hepatocytes is effective in ameliorating superoxide-induced liver injury. Because the metabolism of ethanol and FFAs generate superoxide radicals, we hypothesize that ethanol in the presence of obesity enhances superoxide generation, resulting in enhanced liver injury, and that delivery of SOD in a novel nano- encapsulated form attenuates obesity-associated ALD. The studies proposed in this application will take physiological, pharmacological, and molecular approaches to investigate how reduction of superoxide using nanoSOD will impact the development of ALD with or without obesity. In Specific Aim 1, we will determine the role of nanoSOD in delivering active SOD to hepatocytes and in scavenging ethanol plus LA-induced superoxide in hepatocytes. In Specific Aim 2, we will determine the impact of hepatocyte SOD1 deletion on the progression of ALD with or without obesity and the effectiveness of nanoSOD in ameliorating ethanol and/or obesity-induced ALD. The findings will be relevant to define the role of superoxide in modulating the pathogenesis of obesity-linked ALD and the therapeutic potential of nanoformulated antioxidant enzymes in treating ALD in the presence or absence of obesity.